Background: Since the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak, different animal species have been implicated as possible intermediate hosts that could facilitate the transmission of the virus between species. The detection of these hosts has intensified, reporting wild, zoo, farm, and pet animals. The goal of this study was to determine the seroprevalence of anti-SARS-CoV-2 immunoglobulins (IgG) in domestic dogs and cats and its epidemiological association with the frequency of coronavirus disease 2019 (COVID-19) patients in Villavicencio, Colombia.Methods: 300 dogs and 135 cats were randomly selected in a two-stage distribution by clusters according to COVID-19 cases (positive RT-qPCR for SARS-CoV-2) within the human population distributed within the eight communes of Villavicencio. Indirect enzyme-linked immunosorbent assay (ELISA) technique was applied in order to determine anti-SARS-CoV-2 IgG in sera samples. Kernel density estimation was used to compare the prevalence of COVID-19 cases with the seropositivity of dogs and cats.Results: The overall seroprevalence of anti-SARS-CoV-2 IgG was 4.6% (95% CI=3.2-7.4). In canines, 3.67% (95% CI=2.1-6.4) and felines 6.67% (95% CI=3.6-12.18). Kernel density estimation indicated that seropositive cases were concentrated in the southwest region of the city. There was a positive association between SARS-CoV-2 seropositivity in pet animals and their habitat in Commune 2 . Spearman's correlation coefficients were weakly positive (p=0.32) between the ratio of COVID-19 cases in November 2020 and the results for domestic dogs and cats from the eight communes of Villavicencio.Conclusions: In the present research cats were more susceptible to SARS-CoV-2 infection than dogs. This study provides the first positive results of anti-SARS-CoV-2 ELISA serological tests in domestic dogs and cats in Colombia with information about the virus transmission dynamics in Latin America during the COVID-19 pandemic. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was the causal agent of this disease, which was declared as pandemic by the World Health Organisation (WHO) on 11th March 2020. The zoonotic origin of COVID-19 has been evidenced thanks to the high genomic similarity of SARS-CoV-2 with coronaviruses (CoV) recovered from bats and pangolins, the latter considered potential intermediate hosts of the virus. Betacoronaviruses such as SARS-CoV-2 belong to the family Coronaviridae. They exhibit linear single-stranded RNA of positive polarity and cause respiratory and gastrointestinal diseases in mammals. Even though humans are the most frequent route of transmission, it has been reported that cats (Felis catus) and dogs (Canis lupus familiaris) are susceptible to SARS-CoV-2 infection. This way, reverse zoonosis (anthropozoonosis) is viable through close contact with owners during acute infections.Coronavirus disease 2019 (COVID-19) emerged in the Huanan Seafood Wholesale Market in Wuhan, China, in December 2019. This is the reason why the determination of these hosts has intensified, evidencing a number of reports involving wild, zoo, farm and pet animals. The zoonotic nature from which the transmission hypothesis has started, determines the importance of investigating animal species considered natural reservoirs of SARS-CoV-2. However, concern for the control and reduction of the spread of the virus has led to more vigorous investigation of the role that pet animals, such as dogs and cats, play in the spread of the disease. Although a cat-to-human transmission case was reported, it has been clearly defined that domestic canines and felines do not play a relevant role in the virus transmission to humans.,Since the beginning of the SARS-CoV-2 outbreak, different animal species have been implicated as possible intermediate hosts that could facilitate the virus transmission between species., This fact generates the need to investigate the implications for public and animal health, taking into account that animals are an epidemiological part of this pandemic. Various epidemiological and experimental studies, through serological detection of antibodies against SARS-CoV-2, neutralising antibodies, and detection of viral genome by reverse transcriptase polymerase chain reaction (RT-qPCR), have confirmed SARS-CoV-2 in pet animals around the world. Likewise, the occurrence of emerging variants has been described, as well as their influence on animals, for example, the Alpha variant (B.1.1.7) in dogs and cats with clinical signs of myocarditis, and the Delta variant (B.1.617.2) in dogs with clinical digestive and respiratory symptoms. Regarding the Omicron variant (B.1.1.529), concluded that the SARS-CoV-2 virus accumulated mutations within host cells in mice, giving rise to the Omicron variant that was transmitted to humans, indicating a ‘ping-pong’ (spillover and spillback) evolutionary trajectory between species. Transmission of SARS-CoV-2 Delta variant (AY.127) from hamsters to humans and animal-to-human transmission of SARS-CoV-2 within mink farms have also been reported.On the other hand, human-animal transmission has been widely reported. Canines have lower susceptibility to SARS-CoV-2 infection in contrast to felines, that exhibit greater respiratory pathology and efficient transmission of the virus to other felines through aerosols.Susceptibility to SARS-CoV-2 is determined by the affinity between the receptor-binding domain (RBD) of the viral spike (S) glycoprotein and the angiotensin-converting enzyme 2 (ACE2) of the host cell. Therefore, since vertebrates have conserved domains of ACE2, transmission of the virus between species becomes possible.st May 2022, which reported 676 outbreaks in animals affecting 23 different species in 35 countries, the need for seroepidemiological monitoring in pet, wild and synanthropic animals becomes essential in order to broadly understand the adaptation, evolution and transmission of SARS-CoV-2.In the context of the rapid evolutionary trajectory between species that SARS-CoV-2 has been developing, and taking into account the Report No. 13 of the World Organisation for Animal Health (OIE) of 31 However, seroepidemiological studies of exposure to SARS-CoV-2 by pet animals have not been reported to date in the country. Since the dissemination of SARS-CoV-2 in dogs and cats is weak and short-lived, anti-SARS-COV-2 antibody detection studies are the best choice to determine the circulation of this virus in these companion animals. Of course, the indirect ELISA screening tests that detect immunoglobulin anti-RBD S1 SARS-CoV-2 are called for their greater accuracy in diagnosis. The goal of the present study was to determine the seroprevalence of anti-SARS-CoV-2 immunoglobulins (Ig) class G (IgG) in domestic dogs and cats and its epidemiological association with the frequency of COVID-19 patients in Villavicencio city, Colombia.In Colombia, in December 2021, a lion exhibited symptoms of infection days after being in contact with a COVID-19 positive keeper.This research was endorsed by the Bioethics Committee of the Universidad de los Llanos, according to Minute 02 by consensus of April 6, 2021. In addition, all the owners of the dogs and cats involved in this study signed the respective informed consent.et al. as a reference of p, with SARS-CoV-2 seroprevalences of 3.3% in dogs and 5.8% in cats from Italy. The population assessed in the present study corresponded to 68,651 domestic canines and felines in Villavicencio , according to estimates from the report on anti-rabies vaccination of dogs and cats in Colombia. The confidence interval (CI) considered was 95% and the ‘Z’ value was 1.96 (1-α). The absolute precision considered was 0.15% (d = 0.0051).This is a cross-sectional epidemiological study conducted in Villavicencio, Colombia. It consisted of applying a characterisation survey and taking blood samples from domestic canines and felines. The sample was estimated using the formula for size by proportions in finite populations, using the results obtained by Patterson (according tohttps://www.cdc.gov/epiinfo/esp/es_index.html). The inclusion criteria considered domestic dogs and cats that had lived constantly in their homes for a minimum of two months before starting the present study. We determined as exclusion criteria, the animals that had consumed immunomodulatory medication  one week before the sampling were not included in the study.The participants were selected based on their mandatory participation in the 2021 rabies vaccination campaign, carried out in Villavicencio  by the health secretary. A probabilistic sampling was conducted by randomly selected two-stage clusters of domestic dogs and cats from the eight communes (subdivisions) that compose the urban area of Villavicencio , which cA total of 435 blood samples were taken (300 domestic canines and 135 domestic felines). For this purpose, the authors of this study collected the blood from the jugular or cephalic vein, previous disinfection of the area with alcohol using a 21-gauge needle or vacutainer. Haemostasis was facilitated by applying pressure with sterile gauze in the sampling site for approximately 30 sec. The samples were centrifuged at 2000 g (Centrifuga Eppendorf 5424R) within three hours after being taken, and the sera were stored at -20 °C until analysis in a freezer (ABBA CVANF502B1). The characteristics of each pet recorded were: name; sex; age; species; breed; and owners’ names. This survey also inquired whether the individuals that cohabited with the pet animals (spontaneous communication) had histories of positive or negative RT-qPCR testing for COVID-19, and one of the survey questions was focused on the possibility for the owner to recognize if whether there were histories of clinical signs of the animals, such as signs in the upper or lower respiratory tract, or non-specific digestive signs . Coordinates of the houses where the pets lived, were also recorded. The survey can be found asExtended data.Pet animals characterization was performed through a survey applied to the owners, following the model of a SARS-CoV-2 study that involved dogs and cats with COVID-19 patients in a metropolitan area.® SARS-CoV-2, double antigen multi-species ) according to the manufacturer's instructions. For cats, the kit presents 63% of sensitivity and 96% of specificity. For dogs, the kit has 36% of sensitivity and 85% of specificity. Previous papers used the ID Screen® kit in their studies.– Each plate contained 96 microwells sensitised with recombinant antigen of purified N protein of SARS-CoV-2, to which the following items were added: two negative controls (NC); two positive controls (PC); and 92 problem sera previously homogenised by vortexing. The optical density (OD) reading was performed using the Cytation 3 multimodal microplate reader  with a wavelength of 450 nm. In total, 435 problem sera samples and 25 pre-pandemic canine sera previously stored at -20 °C were analysed. Using the OD data of each well, the sample/positive control (S/P) ratio was calculated, which was expressed as a percentage using the following formula:IgG antibodies against the nucleocapsid protein (N) of SARS-CoV-2 in the sera of domestic dogs and cats were qualitatively determined using the indirect enzyme-linked immunosorbent assay (ELISA) .The step-by-step protocol of this trial has been deposited under the title: Immunoassay of SARS-CoV-2 in dogs and cats V.1, DOI:The frequencies of the data obtained in the survey and transformation of quantitative variables into categories for their subsequent analysis were estimated. The punctual seroprevalence (P) of SARS-CoV-2 in pet animals in Villavicencio was expressed as a proportion using the following formula, considering 95% CI:R 4.2 software with the packages dplyr, MASS, corrplot and epiDispaly, and the maps using theQGIS 3.10 software.The risk association measure odds ratio (OR), calculated by the binomial logistic regression model with 95% CI; was used in order to determine whether the frequency of active COVID-19 cases in humans by commune was related to SARS-CoV-2 seropositivity (exposure) of pet animals. Likewise, Spearman correlation was used to establish a possible relationship in the increase of cases in domestic animals at homes with COVID-19. Finally, using Kernel density analysis, the prevalence of COVID-19 in humans by commune was compared to anti-SARS-CoV-2 IgG seropositivity in domestic dogs and cats. A confidence level of 95% was used for all statistical calculations. Statistical estimates were made using the Specifically, in canines the results indicated 3.67% (95% CI = 2.1-6.4), and in felines 6.67% (95% CI = 3.6-12.18) .-12.18) . Twenty -12.18) . Additio-12.18) .Regarding the eight communes, there was a general seroprevalence of 0, 13.63%, 6.67%, 8.06%, 1.02%, 4.76%, 4.82% and 2.5%, respectively . In the p >0.05) between anti-SARS-CoV-2 IgG seropositivity and the other items assessed . On the other hand, no significant statistical association was found (ssessed . Additiop = 0.32 was found between the ratio of COVID-19 positive cases (RT-qPCR testing) of humans in November 2020 and domestic dogs and cats from the eight Villavicencio communes, result classified as a positive weak correlation. Finally, a strong positive correlation of 0.81 was found between the feline species and their SARS-CoV-2 seropositivity, as well as a positive correlation of 0.68 between the canine species and their SARS-CoV-2 seropositivity  and in felines 6.67% (9/135). Felines had more risk of becoming infected with SARS-CoV-2 that dog   this ten given that there is a weakened immune system, underlying chronic diseases, multiple drug therapies, lack of attention and self-care, poor environmental hygiene, loneliness, and lack of adequate support from other family members in this population. These reasons could be considered with equal value in the case of animals, especially pet animals. On the other hand, Shiet al. reported that three-month-old canines exhibited low susceptibility to experimental infection, contrary to the results obtained in cats, since animals aged less than 100 days and up to nine months were highly susceptible to SARS-CoV-2 infection.As a result of the present study, a positive relationship between seropositivity and the age of the animals was observed.The older animals between 11 and 15 years exhibited this tendency predisposition, but it was not statistically different  . In thisX2 = 0.8206;p = 0.365)  . These r it has been reported with the highest number of inhabitants per house (6 inhabitants) in comparison to the other communes, suggesting that having more than one individual infected with SARS-CoV-2 in the same household increased the risk of infection in these pet animals. Likewise, this commune presented a positive association between the seropositivity of the animals sampled  , which may be related to the duration of the pets’ exposure to the infected owners, and the close contact of the felines with their owners, suggesting the development of antibodies in domestic animals as a consequence of viral transmission from owners.,, In the present study, the association between positive COVID-19 cases (RT-qPCR testing) in humans versus seropositivity in canines and felines from the eight communes of Villavicencio was weakly positive  , which i-30.88)  and the  In the present study, a human-animal transmission was considered based on the results of Smithet al. in the United Kingdom, who ruled out that dogs and cats were reservoirs of infection for humans. However, we cannot be sure about the trasmission direction, which will only be confirmed through further studies. Ultimately, successful elimination of SARS-CoV-2 will only be possible by assessing and controlling transmission in all susceptible animal species, a one health approach that could prevent the re-emergence of the virus in the future., Although the 20 canine pre-pandemic sera reacted negatively to the immunodiagnosis, cross-reactions with ancestral coronaviruses in canines and felines are possible, but in low probability when are compared to commercial ELISAs with neutralizing antibody tests., The possible cross-reactivity and the need to verify if the reactive antibodies are neutralizing for SARS-CoV-2 are the main limitations of our study; likewise, it is advisable to carry out studies of “SARS-CoV-2 virus neutralization test from the serum bank obtained, since this is the gold-standard test for the Centers for Disease Control and Prevention (CDC).Animals and humans are susceptible to a large number of different coronaviruses, in fact, it has been shown that all pathogenic human coronaviruses have their origin in animals, which is why studies should focus on their role in the transmission of SARS-CoV-2.,, The present study does not provide evidence that domestic canines and felines are sources of infection for humans; however, further studies focused on one health should not be ruled out in order to improve our knowledge about transmission, epidemiology and dynamics of SARS-CoV-2 and promote a better response to possible future pandemics.The present study provides the first positive results of anti-SARS-CoV-2 serological tests (ELISA) in domestic dogs and cats in Colombia, with information about the dynamics of virus transmission in Latin America and the world during the COVID-19 pandemic. As mentioned above, cats were more susceptible to natural SARS-CoV-2 infection than dogs, following similar dynamics described in other studies.https://doi.org/10.6084/m9.figshare.21271137.v2.Figshare: Seroprevalence of exposure to SARS-CoV-2 in domestic dogs and cats and its relationship with COVID-19 cases in the city of Villavicencio, Colombia.-Dataset.xlsx , data on domestic dogs and cats that participated in the project.)This project contains the following underlying data:https://doi.org/10.6084/m9.figshare.21271137.v2.Figshare: Seroprevalence of exposure to SARS-CoV-2 in domestic dogs and cats and its relationship with COVID-19 cases in the city of Villavicencio, Colombia.-Consent form.docx-Questionnaire.docxThis project contains the following extended data:Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of the The authors have presented a prevalence study assessing the exposure to SARS-CoV-2 in domestic animals and its association to the COVID19 cases of their human owners, across 8 territorial entities in the city of Villavicencio, Colombia. The work is clearly presented and the authors cite relevant current literature. The study design is appropriate and technically sound; however, I suggest to clarify specifically what is the sample size estimated, and specify if it was estimated per commune. In the Sample size section in Methods the population assessed is referring to the anti-rabies vaccination campaign  and the formula used and parameters but it does not state the estimated “n” for this study. In the Statistical analysis section, please specify how the confidence intervals were calculated. I suggest to the authors to complement in the Methods section the minimization of bias, if it was considered before the study commence. For example, blinding test operators to the positivity status of the owners, selection of the test. Regarding the discussion please do not repeat all the results text again, and do not refer to the Table X or Figure Y, it is not necessary to duplicate it, that information is already detained in the Results section. The authors highlighted several times relationships or associations in comparisons that showed to be “not statistically significantly different”. It is not clear why, it seems they focus on the point estimates and do not consider the confidence intervals. I suggest these statements are based on the statistical measures, in that case, stating that there was not a statistical significant association or please state clearly how do you support your conclusions against the statistical evidence. Keeping in mind what the authors have highlighted, that this study is not assessing the transmission direction between pets and owners, the authors should be careful in the Discussion, Conclusion and Abstract sections, avoiding the statements such as that the evidence from this study show the dynamics of virus transmission. As it is not the case, I suggest to the authors to rephrase such sentences to, for example, the evidence show the association between sero-positivity in pets and COVID-19 human cases in owners. I suggest to the authors to complement in the Discussion section the specific limitations of the study , this will enrich their discussion. For example, the selection of the tests, and its performance, because it seems that the performance declared by the manufacturer of the selected test is much higher for cats than for dogs and this could impact the results of this study.“if whether” in Methods“Dis” in Table 3“Ig” but it refers to IgG in Figure 2Spanish words in Table 4  Typos:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Diagnostics, Immunodiagnostics, Molecular Diagnostics, Research and Development of Diagnostics, Clinical Research of Diagnostics and Medical devices.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The authors have incorporated my previous suggestions into the revised manuscript. I have a minor comment: content of table 4 in the current version of the manuscript is in Spanish.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Human health; molecular biologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. A better description of the rationale of the main aim of the study is needed.It is not clear, in the manuscript, whether the authors examined directly some of the animals, for key clinical signs.The statistical analysis  in table 2 is not clear.Data for dogs in Figure 3 is still not clear.Data for correlation analyses should be presented in the manuscript (or in a supplementary file).In the Discussion section, authors must really keep into account the lack of statistical significance of some findings.The authors took into account several of my previous comments (and it is evident in the revised manuscript). However, several minor aspects highlighted for me in my previous review remain to be considered in the revised manuscript:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Human health; molecular biologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. 1. A better description of the rationale of the main aim of the study is needed. Change accepted: R/ In the last paragraph introduction:Since the dissemination of SARS-CoV-2 in dogs and cats is weak and short-lived, anti-SARS-COV-2 antibody detection studies are the best choice to determine the circulation of this virus in these companion animals. Of course, the indirect ELISA screening tests that detect immunoglobulin anti-RBD S1 SARS-CoV-2 are called for their greater accuracy in diagnosis. Colombia, in December 2021, a lion exhibited symptoms of infection days after being in contact with a COVID-19 positive keeper. 28 However, seroepidemiological studies of exposure to SARS-CoV-2 by pet animals have not been reported to date in the country. Reiners, N, Schnurra, C, Trawinski, H, Kannenberg, J, Hermsdorf, T, Aebischer, A, et al. Performance of a SARS CoV-2 antibody ELISA based on simultaneous measurement of antibodies against the viral nucleoprotein and receptor-binding domain. Eur J Clin Microbiol Infect Dis. (2021) 40:2645–9. doi: 10.1007/S10096-021-04284-5/TABLES/32. is not clear, in the manuscript, whether the authors examined directly some of the animals, for key clinical signs. Change accepted: R/ In the last paragraph subtitle “Sampling and inclusion criteria”one of the survey questions was focused on the possibility for the owner to recognize if whether there were histories of clinical signs of the animals, such as signs in the upper or lower respiratory tract, or non-specific digestive signs  This survey also inquired whether the individuals that cohabited with the pet animals (spontaneous communication) had histories of positive or negative RT-qPCR testing for COVID-19, and3. The statistical analysis  in table 2 is not clear. R/ To determine the individual significance of each of the predictors introduced in a logistic regression model, the Z statistic and the Wald chi-test are used. Databases are included as a supplement that can be used to corroborate the data. At the epidemiological level, the confidence interval offers more information than the p value.4. Data for dogs in Figure 3 is still not clear. Change accepted: R/ Figure 3 will be change.5. Data for correlation analyses should be presented in the manuscript (or in a supplementary file). Change accepted: R/ The data are included as Table 4 in the article.6. In the Discussion section, authors must really keep into account the lack of statistical significance of some findings. R/ Change accepted:p = 0.32). This significance may be influenced by the characterization of the survey, where due to social fear or ignorance some owners could indicate that they were not or had not been positive for the disease, while a strong positive relationship was expected, as in the case of the study conducted by Patterson et al.29 in Italy. On the other hand, the study by Van Aart et al.43 showed that none of the felines had been infected with SARS-CoV- despite the fact that these were living with their positive COVID-19 owners.  Therefore, these associations between species should be analysed considering different factors.” “In the present study, the association between positive COVID-19 cases (RT-qPCR testing) in humans versus seropositivity in canines and felines from the eight communes of Villavicencio was weakly positive " is not clear (it is not clear that it is additive).In "Villavicencio (Meta State)". "state" is not clear, in the context of the organization of Colombia.A better description of the recruitment strategy for the current study would be useful.In Methods, a better description of "commune" is needed.It is not clear whether the authors examined directly some of the animals, for key clinical signs.Key parameters, such as specificity and sensitivity, are not described for the ELISA assay used. In addition, in Methods it would be important to include citations to key previous papers using the same kit.In Statistical analysis, a description of the specific R packages used is needed.Table 2 is incomplete (the header for a group of rows is missing) and the statistical analysis in table 2 is not clear.Figure 2 might be presented as supplementary information.Figure 3 is not clear - it is not clear what is the signal for humans and what is the signal for animals; in addition authors might use colors in this figure.Data for correlation analyses should be presented.In the Discussion section, authors must keep into account the lack of statistical significance of some findings.Possible cross-reactivity of the ELISA assay is not discussed in the manuscript.Limitations of the current study and recommendations for future studies are needed.The manuscript describes an analysis of seroprevalence of anti-SARS-CoV-2 IgG in a sample of 300 dogs and 135 cats from a city in Colombia. Several major and minor issues should be addressed:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Human health; molecular biologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. 1. A better description of the rationale of the main aim of the study is needed - why are you studying only seroprevalence? R/ One of the main limitations of our study is the lack of economic support. We could have done more advanced studies such as seroincidence, detection of neutralizing antibodies, or isolation of viral genomic material. Nevertheless, we just study seroprevalence in order to include an important and representative sample size of cats and dogs in Villavicencio. 2. "SARS-CoV-2 seroprevalence of 9.1% (3.3% in canines and 5.8% in felines)" is not clear (it is not clear that it is additive). R/ Change accepted:MethodsType of study and sample size paragraphet al.29 as a reference of p, with SARS-CoV-2 seroprevalences of 3.3% in dogs and 5.8% in cats from Italy”. Line 2 “The sample was estimated using the formula for size by proportions in finite populations, using the results obtained by Patterson 3. In "Villavicencio (Meta State)". "state" is not clear, in the context of the organization of Colombia. R/ Colombia is divided into 32 departments and one capital district (Bogotá). Each Department has a capital city. In the case of the Meta Department, the capital city is Villavicencio. Change accepted: The word “State” will be changed for Department on each occasion.Methods E.g.Sampling and inclusion criteria paragraph Line 1 “The participants were selected based on their mandatory participation in the 2021 rabies vaccination campaign, carried out in Villavicencio  by the health secretary”. However, in some publications the word “state” means “departamento”. 4. A better description of the recruitment strategy for the current study would be useful. R/ The inclusion and exclusion criteria used in order to estimate the sample size are mentioned under the subheading “Sampling and inclusion criteria”. Change accepted: The subheading will change to “Sampling, inclusion, and exclusion criteria” The sentence “The animals that had consumed immunomodulatory medication  one week before the sampling were not included in the study.” Will be changed for: “We determined as exclusion criterion, the animals that had consumed immunomodulatory medication  one week before the sampling.” 5. In Methods, a better description of "commune" is needed. R/ In Colombia, Departments are subdivided into municipalities which are in turn subdivided into “corregimientos” in rural areas and into “comunas” (communes) in urban areas. Each commune, in turn is divided into neighborhoods. Change accepted:MethodsSampling and inclusion criteria paragraph Line 2 “A probabilistic sampling was conducted by randomly selected two-stage clusters of domestic dogs and cats from the eight communes (subdivisions) that compose the urban area of Villavicencio , …”. 6. It is not clear whether the authors examined directly some of the animals, for key clinical signs. R/ No, animals were randomly selected among those who participated in the rabies vaccination campaign. Once these were selected, a survey was applied, and one of the questions was related to the clinical signs. 7. Key parameters, such as specificity and sensitivity, are not described for the ELISA assay used. In addition, in Methods it would be important to include citations to key previous papers using the same kit. R/ Change accepted:MethodsImmunoassay paragraph® SARS-CoV-2, double antigen multi-species ) according to the manufacturer's instructions. For cats, the kit presents 63% of sensitivity and 96% of specificity. For dogs, the kit has 36% of sensitivity and 85% of specificity. Previous papers used the ID Screen® kit in their studies (48-51). Line 1. IgG antibodies against the nucleocapsid protein (N) of SARS-CoV-2 in the sera of domestic dogs and cats were qualitatively determined using the indirect enzyme-linked immunosorbent assay (ELISA) .PubMed Abstract IPublisher Full Text 48. Bessière P, Vergne T, Battini M, Brun J, Averso J,et al.:A comparative study of eight serological methods shows that spike protein-based ELISAs are the most accurate tests for serodiagnosing SARS-CoV-2 infections in cats and dogs.Front. Vet. 2023. Sci.10(1121935).Publisher Full Text 49. Diezma-Díaz C, Álvarez-García G, Regidor-Cerrillo J, Miró G, Villanueva-Saz S,et al.:Antibodies to SARS-CoV-2 in dogs and cats, USA.Emerg Microbes Infect. 2021.10(1):1669-1674.PubMed Abstract IPublisher Full Text 50. Barua S, Hoque M, Adekanmbi F, Kelly P, Jenkins-Moore M,et al.:A household case evidences shorter shedding of SARS-CoV-2 in naturally infected cats compared to their human owners.Emerg Microbes Infect. 2021.10(1):376-383.PubMed Abstract IPublisher Full Text 51. Neira V, Brito B, Agüero B, Berrios F, Valdés V, 8. In Statistical analysis, a description of the specific R packages used is needed. R/ Change accepted:MethodsStatistical analysis paragraph Line 9. Statistical estimates were made using the R 4.2 software with the packages dplyr, MASS, corrplot and epiDispaly, and the maps using the QGIS 3.10 software. 9. Table 2 is incomplete (the header for a group of rows is missing) and the statistical analysis in table 2 is not clear. R/ In theTable 2, the header of the row 1, 2, 3, 4 … 8: is “Communes”. This header is included in the original manuscript. On the other hand, statistical analysis included in table 2 are described in the fourth paragraph of results: Line 1 “Regarding the SARS-CoV-2 exposure and the risk factors analysed, a statistically significant association between SARS-CoV-2 seropositivity and Commune 2 was found .” 10. Figure 2 might be presented as supplementary information. R/ Change not accepted, it is important to show the OD distribution among communes, showing the positive results and grey area results. 11. Figure 3 is not clear - it is not clear what is the signal for humans and what is the signal for animals; in addition authors might use colors in this figure. R/ Change accepted:Results Figure 3 will be change by the colored one.  12. Data for correlation analyses should be presented. R/ Change not accepted, here we present a table with the data for the correlation analyses about the association between positive COVID-19 cases (RT-qPCR testing) in humans versus seropositivity in pets. Nevertheless, it is not necessary to show the table in the paper, since we mention the important data in the discussion .                       Human   Dogs    Cats    Pets Dogs                0,01         1         --          -- Cats                0,33       0,17      1 Pets                 0,32      0,68    0,81*       1p-value = 0.01467 * 13. In the Discussion section, authors must keep into account the lack of statistical significance of some findings. R/ In the results and discussion sections, we mentioned and pointed out that results were not significant ; however, there were trends that correlate with other studies and we mentioned them explaining that these were not significant :but it was not statistically different”. “As a result of the present study, a positive relationship between seropositivity and the age of the animals was observed. The older animals between 11 and 15 years exhibited this tendency predisposition, 14. Possible cross-reactivity of the ELISA assay is not discussed in the manuscript. R/ Change accepted: The following paragraph will be added to the discussion section.Discussion  Although the 20 canine pre-pandemic sera reacted negatively to the immunodiagnosis, cross-reactions with ancestral coronaviruses in canines and felines are possible, but in low probability when are compared to commercial ELISAs with neutralizing antibody tests .et al.:Evaluation of a SARS-CoV-2 surrogate virus neutralization test for detection of antibody in human, canine, cat, and hamster sera.J Clin Microbiol. 2021;59(2):e02504–20. PubMed Abstract IPublisher Full Text 54. Perera R, Ko R, Tsang O, Hui D, Kwan Met al.:SARS-CoV-2 serosurveillance in a patient population reveals differences in virus exposure and antibody-mediated immunity according to host demography and healthcare setting.J Infect Dis. 2021;223(6):971-980. PubMed Abstract IPublisher Full Text 55. Hughes EC, Amat JAR, Haney J, Parr J, Logan N, 15. Limitations of the current study and recommendations for future studies are needed. R/ Change accepted: The following paragraph will be added to the discussion section.Discussion  The possible cross-reactivity and the need to verify if the reactive antibodies are neutralizing for SARS-CoV-2 are the main limitations of our study; likewise, it is advisable to carry out studies of SARS-CoV-2 neutralization from the serum bank obtained, since this is the gold-standard test for the Centers for Disease Control and Prevention (CDC) (56). Coronavirus disease 2019 (COVID-19) 2020 interim case definition, approved April 5, 2020.2023 [cited 2023 February 9].  Reference Source 56. Centers for Disease Control and Prevention.


Introduction: The evidence for remdesivir therapy in immunocompromised patients is scarce. To evaluate remdesivir (RDV) effectiveness and safety in COVID-19 outpatients at high risk for progression in a real-world setting, we compare the outcome in immunocompromised (IC) patients with that in non-immunocompromised patients.Methods: Two hospitals conducted a retrospective study of all adult patients with mild-to-moderate SARS-CoV-2 infection at high risk for disease progression who were treated as outpatients with a 3-day course of RDV (1st January−30th September 2022). The primary effectiveness endpoint was a composite of any cause of hospitalization or death by day 30. A multiple logistic regression model was built to explore the association between immune status and clinical outcome, estimating adjusted odds ratios [aORs (95% CI)].Results: We have included 211 patients, of which 57% were males, with a median age of 65 years (IQR 53–77), 70.1% were vaccinated (three or four doses), and 61.1% were IC. The median duration of symptoms before RDV treatment was 3 days (IQR 2–5). During follow-up, 14 (6.6%) patients were hospitalized, of which 6 (2.8%) were hospitalized for COVID-19 progression. No patient required mechanical ventilation, and two patients died (non-COVID-19-related). After accounting for potential confounders, only anti-CD20 treatment was associated with the composed outcome [aOR 5.35 ], whereas the immunocompetence status was not [aOR 1.94 ].Conclusion: Early COVID-19 outpatient treatment with a 3-day course of remdesivir in vaccinated patients at high risk for disease progression during the Omicron surge had a good safety profile. It was associated with a low rate of all-cause hospitalization or death, regardless of immunocompetence status.  Antiviral treatment  for non-hospitalized COVID-19 patients at high risk of progression was initially supported by clinical trials in unvaccinated people during the pre-Omicron wave . SubsequRemdesivir has been associated with reduction or progression in hospitalized patients who required no or conventional oxygen support, according to a recent meta-analysis . HoweverWe assessed the effectiveness and safety of RDV in high-risk outpatients with mild-to-moderate COVID-19 and whether immunocompetence status could influence the outcome.This is a retrospective real-world study conducted in two hospitals in Spain  and Vega Baja Hospital of Orihuela (HVB), Alicante). We included adult patients with mild-to-moderate SARS-CoV-2 infection (confirmed by RT-PCR) at high risk for disease progression, who were treated as outpatients with a 3-day course of RDV, from 1st January to 30th September 2022.2), chronic pulmonary disease, neurocognitive disorders and chronic cardiovascular and renal diseases.Treatment was initiated according to the treatment criteria established by The Spanish Agency for Medicine and Health Products (AEMPS) during the study period. Treatment was initiated in outpatients with mild-to-moderate infection in the first 7 days of symptom onset and stratified into one of the following categories: 1) all unvaccinated patients aged >65 years; 2) immunocompromised (IC) patients; and 3) patients aged >65 years, vaccinated >180 days after the last dose with at least one of the following risk factors for disease progression: diabetes mellitus, with complications, obesity  for data management. The variables at HVB were extracted from electronic medical records. In both hospitals, the variables were recorded by expert clinicians, thereby minimizing information bias. The main explanatory variable was immunocompetence status. All patients were followed for a minimum of 30 days.The demographic and clinical data at HGUA were obtained from the structured clinical registry developed using the The primary effectiveness endpoint was a composite of any cause of hospitalization or death by day 30 (excluding those produced in the first 24 h of starting treatment). The cause of hospitalization and mortality was registered. The safety endpoint was any adverse event.Categorical variables are expressed as frequencies and their percentages (%). Continuous variables are expressed through means and standard deviations or medians and interquartile range (IQR), depending on whether or not they follow a normal distribution. The index date was the outpatients’ COVID-19 clinical assessment date, and the final follow-up date was 30 days later, unless previously censored.p-values below 0.100 and were considered main confounding factors such as age [≤80/>80], gender [male/female], and IC status [IC/non-IC]).Multiple logistic regression models were built to explore risk factors associated with hospitalization or fatal outcome; adjusted odds ratios (aOR) with 95% confidence intervals (95% CI) were estimated. Variables were included as covariates if they showed associations in simple models with p < 0.050 was defined as statistically significant.The statistical analysis of the results was carried out using the IBM SPSS Statistics version 22 program . A A total of 393 patients were treated during the study period, of which 211 patients were included in the analysis  underwent solid organ transplant, 20 (15.5%) and 19 (14.7%) had ongoing chemotherapy for cancer, 16 (12.4%) underwent anti-CD20 therapy, 16 (12.4%) were given other biologic immunosuppressive drugs, 10 (7.0%) were given non-biological immunosuppressive drugs, and 21 (16.3%) had other immunosuppressive conditions .The primary effectiveness endpoint was met by 196 patients. Fourteen (6.6%) patients were hospitalized and two died (0.9%) (the deaths of both patients were considered unrelated to COVID-19). Six patients (2.8%) were admitted for COVID-19-related pneumonia. No one required mechanical ventilation.Nine of 129 patients (7%) in the IC group and five of 82 (6.1%) in the non-IC group had been hospitalized by day 30 ; COVID-19-related hospitalization rates were 3.1% and 2.4%, respectively .p-value < 0.100 , gender, age, and IC status. Treatment with anti-CD20 was independently associated with the combined outcome  , whereas a duration of more than 4 days from symptom onset to RDV infusion  was close to signification , gender, age, and IC status. Treatment with anti-CD20 was not associated with non-death hospitalization  during the Omicron variant wave. The immunocompetence status, as a global risk factor, does not seem to influence the response to RDV treatment. Only treatment with anti-CD20 was independently associated with hospitalization and death in this population treated early with RDV.Both vaccination and the Omicron variant have been associated with a more benign course, with hospitalization rates, without treatment, of 0.5%–3.2% (in large published cohort studies), , but theEvidence for RDV therapy in improving clinical outcomes in non-hospitalized patients with moderate-to-severe COVID-19 is scarce. The rates of hospitalization or death in the only outsetting clinical trial of RDV (PINETREE),  were 0.7N = 126) reported a significant reduction in hospitalization or death [aHR 0.16 (95% CI 0.06–0.44)]. Recently, several small observational studies of RDV treatment for IC outpatients have been published by In these reports, hospitalization for any cause has been used as the main outcome. However, in patients with complex medical disorders, hospitalizations related to underlying condition, and not to COVID-19, are common, hindering the interpretation of the results. A definition based on the number of COVID-19-related hospitalizations or death would be better to assess the effectiveness of treatment. In our cohort, the COVID-19-related hospitalization rate was less than 3%, and no deaths were registered. Although the patients in the IC cohort were younger and vaccinated more frequently, the non-IC group had a higher number of comorbidities; however, the rate of progression was similar after adjusting for confounding factors. Hence, based on our results, early RDV treatment could be as effective in IC patients as in non-IC patients with other high-risk factors.Administration of monoclonal anti-CD20 antibody has been associated with worse clinical outcomes in patients with COVID-19 . In our Some important limitations need to be addressed. First, this was a two-center retrospective observational study, and although an effort was made to control for relevant confounding factors, unmeasured confounding variables may still have been present. Second, this study has no control group . Finally, viral sequencing was not systematically performed; we assume an Omicron variant of SARS-CoV-2 as the dominant circulating strain based on data tracking reported by our center. These limitations were counterbalanced by a large cohort of high-risk patients managed through a clinical pathway, allowing for a comparative analysis of outcomes.In this outpatient real-world study during the Omicron wave, early treatment with a 3-day course of remdesivir had a good safety profile and prevented disease progression regardless of immunocompetence status. These data support another therapeutic option for the management of non-hospitalized vulnerable vaccinated patients who are at high risk for progression to severe COVID-19.


Background: TP53 is the most commonly mutated gene in human cancer with loss of function mutations largely concentrated in “hotspots” affecting DNA binding. APR-246 and COTI-2 are small molecules under investigation in P53 mutated cancers. APR binds to P53 cysteine residues, altering conformation, while COTI-2 showed activity in P53 mutant tumors by a computational platform. We compared APR-246 and COTI-2 activity in human tumor explants from 247 surgical specimens. Methods: Ex vivo analyses of programmed cell death measured drug-induced cell death by delayed-loss-of-membrane integrity and ATP content. The LC50s were compared by Z-Score. Synergy was conducted by the method of Chou and Talalay, and correlations were performed by Pearson moment. Results: APR-246 and COTI-2 activity favored hematologic neoplasms, but solid tumor activity varied by diagnosis. COTI-2 and APR-246 activity did not correlate (R = 0.1028) (NS). COTI-2 activity correlated with nitrogen mustard, cisplatin and gemcitabine, doxorubicin and selumetinib, with a trend for APR-246 with doxorubicin. For ovarian cancer, COTI-2 showed synergy with cisplatin at 25%. Conclusions: COTI-2 and APR-246 activity differ by diagnosis. A lack of correlation supports distinct modes of action. Cisplatin synergy is consistent with P53’s role in DNA damage. Different mechanisms of action may underlie disease specificity and offer better disease targeting. TP53 tumor suppressor is the most commonly mutated gene in human cancer .Eprenetapopt APR-246) is a novel, first-in-class, small molecule that selectively induces apoptosis in ormation ,21,22. I6 is a noormation ,26,27. Eo models . In a pho models ,30. Rece®, which uses a unique combination of traditional and modern pharmacology principles, statistical modelling, medicinal chemistry and machine-learning technologies to discover and optimize novel compounds for the treatment of cancer .To examine the activity of APR-246 and COTI-2, we applied the ex vivo analysis of programmed cell death (EVA/PCD) to 247 human tumor primary culture specimens obtained from surgical procedures, cytologically positive fluids and peripheral blood that were submitted to our CLIA-licensed laboratory. The EVA/PCD platform has been shown to correlate with the response, time to progression and survival in human cancer ,35. The The ex vivo analysis of programmed cell death methodology has previously been described ,37,38. BTP53 mutations were subdivided by the type and location of the mutation.For 49 APR-246 tested and 86 COTI-2 tested samples, genomic data was available as provided by commercial next-generation DNA sequencing (NGS) laboratories, including Foundation One, CARIS, Neo Genomics, Guardant 360, Memorial Sloan-Kettering and MD Anderson, among others. −1 penicillin and 100 μg mL−1 streptomycin (modified RPMI 1640) and were submitted directly to our laboratory. The samples obtained after hours were maintained at 4 °C until processed . Patients undergoing surgical procedures provided sterile tumor specimens for analysis. All the patients provided written consent to allow the use of tumor biopsy specimens. At the time of surgical exploration, the tumor tissues removed from the patients were processed sterilely and placed in sterile RPMI 1640 media containing 2 mm l-glutamine, 15% fetal calf serum, 100 IU mL6 cells mL−1 and distributed into 96-well polypropylene culture plates (90 μL well−1). Serial dilutions of the drugs and drug combinations in 10 μL volumes were added, and the plates were incubated at 37 °C in 6% CO2 in a sterile, humidified environment for 72 h, as described in the next section.The tumor specimens were mechanically disaggregated by sterile mincing with scalpels. The specimens were then incubated for 2 h in 0.8% collagenase IV and 0.002% DNaseI. The cells were isolated by density centrifugation over Ficoll–Hypaque, washed and then resuspended in modified RPMI 1640. The cell suspensions were adjusted to 1 × 10The dose-dependent cytotoxicity of the drugs was investigated using a five-point dose–response curve. Serial dilutions of the drugs were prepared in 0.15 m NaCl, and 10 μL of a drug solution was added to each well. The cells were incubated for 72 h with saline vehicle control (0.15 m NaCl) or in the presence of APR-246 (40.2 to 2.5 μMol), COTI-2 (317 to 19 μMol), cisplatin (22 to 1.4 μMol), doxorubicin (2.2 to 0.14 μMol), gemcitabine (483 to 29 μMol), nitrogen mustard (28 to 1.8 μMol), paclitaxel (35 to 2.2 μMol), everolimus (20.9 to 1.3 μMol), selumetinib (109 to 6.8 μMol), cisplatin and gemcitabine at a fixed ratio (22 to 484 μMol) or cisplatin and COTI-2 at a fixed ratio (22 to 6.8 μMol).After 72 h, 10 μL of phosphate-buffered saline containing 0.5% nigrosin-B and 1% Fast Green and 37,500 glutaraldehyde-fixed avian red blood cells  were added to each well, and the samples were gently agitated to facilitate mixing. After 10 min, the samples were aspirated and then centrifuged onto a glass slide using a modified cytospin cassette. The air-dried samples were counterstained with modified haematoxylin and eosin. The tumor cell viability was determined as the ratio of living tumor cells over simultaneously counted avian red blood cells. The cell survival of the drug-treated samples was expressed as a percentage of the saline control values.The ATP concentration is measured using a luciferin reagent that is converted into oxyluciferin by luciferase, with the number of viable cells in the culture shown to correlate with the luminescence over 3 orders of magnitude . The cell viability is measured after 72 h by introducing 100 μL of CellTiterGlo solution to each well in the 96-well plate. The plates are shaken for 2 min. The cell suspension is then incubated for 10 min. The cell suspension is then shaken for an additional 2 min. The 96-well plate is read on a Fluoroskan Ascent FL instrument, according to the manufacturer’s instructions. The 50% lethal concentration (LC50) values were calculated using a least-squares line of best-fit generated from the five-point concentration curve, with the LC50 values interpolated from the curve. The synergy was determined using the median effect technique . In brieThe overall mean LC50 (LC50T) and s.e.m. for all the tumor types (s.e.m. T) within our laboratory database were utilized to calculate the Z-scores by the following formula:The use of inferential statistics is predicated upon the central limit theorem, a fundamental sampling theorem that states that the distribution of a sample population is unimportant so long as the samples are drawn randomly in sufficient numbers from a parent population whose distribution need not have a normal (Gaussian) distribution .By normalizing the LC50 values for each single agent and/or combination around the mean LC50 values, the samples found to be more resistant than average fell to the right of the mean, whereas those that were more sensitive than average fell to the left of the mean. This Z-score transformation is routinely used in the US by the National Cancer Institute for studies comparing microarray data and other analyses . It has p ≤ 0.05. For comparisons of in vitro activity or combinations, the LC50 values were normalized to Z-scores (with the mean set to 0 and variance from the mean in s.e.m. units of 1). This permitted direct comparisons of the sensitivity versus the resistance of single agents and drug combinations across tumor types. Using these normalized LC50 values, graphic representations were generated, depicting on the left side of the mean those samples that were more sensitive than average to drug combinations (negative range) and on the right side of the mean those that were more resistant than average (positive range). Pearson product moment correlations were used to provide R values, which were examined by a two-tailed Student t-test for significance <0.05. Bonferroni corrections were applied by dividing the 0.05 value by the number of separate correlations conducted.The LC50 means for each agent were compared across the tumor types, with the criterion for statistical significance set at A comparison of disease-specific activity for APR-246 and COTI-2 was conducted by (SEM) Z-Score analyses . As can TP53 measured by next-generation DNA sequencing (NGS)  versus hypomorphic , we conducted a (SEM) Z-Score analysis for COTI-2 and AZD 246 that compared the drug activity with the NGS-measured mutation type . We provng (NGS) .Representative dose–response curves using serial dilutions of APR-246 and COTI-2 to measure drug-induced cytotoxicity are provided a,b. To interrogate the mechanisms of action for COTI-2 and APR-246, we examined correlations between the compounds and other classes of drugs by Pearson moment R value. As can be seen in A final analysis applied the method of Chou and Talalay  to examiTP53 mutations on efficacy using a previously validated 3-D human tumor explant platform.We undertook this analysis to compare and contrast the activity of these two novel compounds and explore the disease specificity, possible mechanisms of action, combinatorial potential and impact of specific Our findings reveal that COTI-2 and APR-246 are cytotoxic to human tumor 3-D explants. Though structurally and mechanistically different, both agents have been developed to target tumors with P53 mutations. When we directly compared the activity for these two agents by Pearson moment, the R value of 0.1 clearly supports distinct mechanisms of action.The activity profiles for both agents favor hematologic neoplasms. Many cytotoxic drugs are more active in blood-borne tumors compared with solid tumors. As only 20% of hematologic tumors carry P53 mutations, yet the activity profiles favor hematologics over solid tumors, even ovarian cancers, where up to 80% of patients carry P53 mutations, the activities for both compounds appear to extend beyond their direct impact upon P53. For APR-246, this may reflect its effect on the redox state via thioredoxin reductase, while COTI-2 appears to have many effects downstream of P53.TP53 mutation type upon drug activity supports APR-246’s putative mode of action as one that alters the conformation of the P53 protein, in as much as only hypomorphic mutations appeared to be sensitive to this compound with virtually no activity for the amorphic subtypes. In contrast, COTI-2 activity was found in both the amorphic and hypomorphic subtypes, suggesting a mode of action that functions downstream of the P53 protein.An examination of the impact of the Exploratory analyses enabled us to examine the synergy for cisplatin combined with COTI-2 in ovarian cancer specimens. This is the subject of a clinical trial examining COTI-2 alone and in combination with cisplatin in gynecologic malignancies (NCT02433626). Based on the putative mechanisms of action of COTI-2 in DNA damage repair, the 25% synergy level was somewhat lower than we had expected.p = 0.02) in a small subset of specimens with amorphic P53 mutations. This will be further examined in additional samples as specimen availability allows.An additional finding was a high correlation between CDDP and COTI-2  platform has been shown to correlate with the response, time to progression and survival, the insights gained from this study may allow for the better selection of treatment candidates both by disease type and individual patient profile and can facilitate the development of effective drug combinations based upon the measurement of potential therapeutic synergy. Validated primary culture techniques have the capacity to measure all the operative cellular mechanisms of drug response and resistance, acting in concert to provide clinically relevant signals for patient therapy and accelerate drug development.


The Tenosynovial giant cell tumor Observational Platform Project (TOPP) registry is an international prospective study that previously described the impact of diffuse-type tenosynovial giant cell tumour (D-TGCT) on patient-reported outcomes (PROs) from a baseline snapshot. This analysis describes the impact of D-TGCT at 2-year follow-up based on treatment strategies.TOPP was conducted at 12 sites . Captured PRO measurements assessed at baseline, 1-year, and 2-year follow-ups were Brief Pain Inventory (BPI), Pain Interference, BPI Pain Severity, Worst Pain, EQ-5D-5L, Worst Stiffness, and Patient-Reported Outcomes Measurement Information System. Treatment interventions were no current/planned treatment (Off-Treatment) and systemic treatment/surgery (On-Treatment). n = 79), BPI Pain Interference (1.00 vs. 2.86) and BPI Pain Severity scores (1.50 vs. 3.00) were numerically favorable in patients remaining Off-Treatment compared with those who switched to an active treatment strategy at year 1. From 1-year to 2-year follow-ups, patients who remained Off-Treatment had better BPI Pain Interference (0.57 vs. 2.57) and Worst Pain (2.0 vs. 4.5) scores compared with patients who switched to an alternative treatment strategy. In addition, EQ-5D VAS scores (80.0 vs. 65.0) were higher in patients who remained Off-Treatment between 1-year and 2-year follow-ups compared with patients who changed treatment strategy. For patients receiving systemic treatment at baseline, numerically favorable scores were seen in patients remaining on systemic therapy at 1-year follow-up: BPI Pain Interference (2.79 vs. 5.93), BPI Pain Severity (3.63 vs. 6.38), Worst Pain (4.5 vs. 7.5), and Worst Stiffness (4.0 vs. 7.5). From 1-year to 2-year follow-up, EQ-5D VAS scores (77.5 vs. 65.0) were higher in patients who changed from systemic treatment to a different treatment strategy.A total of 176 patients (mean age: 43.5 years) were included in the full analysis set. For patients without active treatment strategy (Off-Treatment) at baseline  The TOPP registry is an international prospective study that previously described the impact of diffuse-type tenosynovial giant cell tumor on patient-reported outcomes from a baseline snapshot. This article reports a 2-year follow-up based on treatment strategies and could represent a benchmark for future clinical trials. These findings extend beyond what has been previously published, as this analysis is the first to describe the impact of D-TGCT on PRO as a prospective 2-year follow-up based on treatment strategies and could represent a benchmark for future clinical trials. As optimal treatment strategies remain to be elucidated, the treatment of D-TGCT patients is multidisciplinary and multimodal. The practical use of PRO assessments for treatment evaluation in this rare disease needs to be further evaluated in future studies.1 It affects small and large joints (mainly the knee), and symptoms include pain, stiffness, swelling, and limited range of motion.3 Two subtypes of TGCT are defined based on clinical and radiological characteristics: localized- and diffuse-type TGCT (L-TGCT and D-TGCT).5 Both subtypes share a common pathophysiology, and they represent a wide spectrum of clinical entities, making TGCT behavior complex and hard to predict.6 D-TGCT constitutes 10%–20% of all TGCT cases, usually occurring in large joints , is an aggressive multi-lobulated lesion located intra- and/or extra-articular, and has a detrimental effect on the quality of life (QoL).7-11Tenosynovial giant cell tumor (TGCT) is a rare, locally aggressive mesenchymal neoplasm arising from the synovium of joints, bursae, and tendon sheaths, associated with colony-stimulating factor 1 (CSF1) overexpression.12 The current standard of care for patients with TGCT is surgical resection of the tumor as completely as possible to reduce symptoms and prevent joint destruction, improve function, and minimize the risk of recurrence.13 While surgery cures the vast majority of L-TGCT cases, D-TGCT shows a high tendency toward local recurrence, occurring in over half of the resected cases. Therefore, the value of surgery in the D-TGCT subtype is variable.14-16A proper diagnosis of TGCT can take several years due to the non-specific symptoms and the rarity of this disease, resulting in severely delayed optimal treatment and care and a higher risk of insufficient treatment or undertreatment for these patients.17-19 Pexidartinib has been approved by the US Food and Drug Administration at a dosage of 400 mg twice daily, and the National Comprehensive Cancer Network added it as a category 1 recommendation for the treatment of adult patients with severe symptomatic TGCT associated with severe morbidity or functional limitations and not amenable to improvement with surgery.21 Pexidartinib is only available through a restricted program under a Risk Evaluation and Mitigation Strategy (REMS), because of the risk of hepatotoxicity, and was not approved by the European Medicines Agency. Other non-registered systematic therapies have been used either off-label or in clinical trials .23The CSF1 receptor (CSF1R) has been the target of promising therapies.24 Questions to elaborate the true morbidity and actual impact on QoL of both the disease and its various treatment options remain to be elucidated.To date, most epidemiologic understanding of D-TGCT comes from small, retrospective studies that traditionally focused on oncological outcomes.8Presently, there are few data available detailing the management of patients with TGCT, the disease effect of TGCT for patients  or the economic impact of TGCT. Patient-Reported Outcomes Measurement Information System-Physical Function (PROMIS-PF) was developed in 2005 by the National Cancer Institute (NCI) to measure the impact of the disease on patients’ ability to perform daily activity through self-administered items and validated for patients with TGCT in 2019.25 In addition, a more recent analysis of this registry provided a picture of the treatment journey of D-TGCT patients as a 2-year observational follow-up.26 This analysis is the first to describe the impact of the disease on PROs at a 2-year follow-up based on treatment strategies.Previously, the prospective international TGCT Observational Platform Project (TOPP) registry described the impact of TGCT on patient-reported outcomes (PROs) from a baseline snapshot.25This global multicenter, prospective sponsored study included all consecutive patients from 12 tertiary sarcoma centers in 7 EU countries and 2 US sites. Key eligibility criteria and study designs for the TOPP registry have been described elsewhere.25 While watchful waiting was a common treatment strategy, systemic treatment was preferred for patients with primary disease, whereas patients with recurrent disease often were treated with surgery and other treatment modalities.26 TGCT was histologically confirmed and assessed as diffuse based on magnetic resonance imaging (MRI) or clinical presentation, if MRI was missing. The observation period time per patient was 24 months (± 2 months).Briefly, patients aged ≥18 years had primary or recurrent D-TGCT. Recurrent disease was defined as tumor recurrence after complete resection or progression of the residual tumor.PRO measurements were captured at baseline, 6 months, 1 year, 18 months, and 2 years. For this analysis, PROs taken at baseline (at time of enrollment in the registry), and at the 1-year and 2-year follow-up collection points were used .n = 79). Patients undergoing active intervention  were classified as On-Treatment (n = 97). Of these 97 patients, 84 (either systemic therapy only [n = 45] or surgery only [n = 39]) were included in this analysis. The other 13 patients  were not included as the patient populations were regarded too small to analyze.Patients were followed prospectively, and data were collected based on the type of treatment strategy. Patients who were actively surveilled during the 2-year observation period were not actually being treated and did not have a planned treatment were categorized as Off-Treatment , and if undergoing systemic treatment (at baseline or within the 2-year period). Furthermore, patients undergoing surgery at baseline were also considered “On-Treatment” within the 2-year observation period as surgery does not entail a procedure at each time point .PRO endpoints were calculated as medians of all available scores collected at each time point for each patient. Only patients who filled out the PRO questionnaires at a specific time point  were included in the analysis. The following were assessed:Developed by the Pain Research Group Anderson Cancer Center, University of Texas, the BPI rapidly assesses the severity of pain and its impact on daily functioning. It measures the impact of pain on daily function, location of pain, pain medications, and amount of pain relief in the past 24 h. The BPI gives 2 main scores: a pain severity score and a pain interference score. The BPI Pain Severity scale (rated from 0 = no pain to 10 = pain as bad as you can imagine) is calculated from 4 items about pain intensity; BPI Pain Interference scale  corresponds to the item on pain interference with 7 sub-items. Also, the BPI Worst Pain scale (rated from 0 = no pain to 10 = pain as bad as you can imagine) rates the patient’s pain by circling a number (0-10) that best describes their pain at its worst over the last 24 h.The first part consists of 5 domains . Each domain has 5 levels ranging from “no problems” through “profound difficulties” with a sum utility score ranging from 0 to 1. In the second part, patients have to rate their current health on a 20-cm vertical visual analog scale .A single-item (from 0 to 10 with 0 = no stiffness and 10 = stiffness as bad as you can imagine) numerical rating scale.T-scores, which are standardized to a mean of 50 and a standard deviation (SD) of 10, where a higher score signified better physical function. Calculation of PROMIS scores was performed according to the scoring procedure by the Health Measures Scoring Service.27PROMIS Physical Function (PROMIS-PF) rating scale . More specifically, the results from both sets (upper extremity and lower extremity) were combined and analyzed together. Each item . Scores were expressed as 90Yttrium, systemic treatment + other , or surgery + other treatment .Patients On-Treatment at baseline, followed by the same type of treatment or wait-and-see at years 1 and/or 2 were documented as “remaining on the same treatment.” Treatment interventions at years 1 and/or 2 were classified as Off-Treatment, systemic treatment only , surgery only, radiotherapy or Binary, categorical, and ordinal parameters have been summarized by means of absolute and percentage numbers within the various categories . Numerical data were summarized using standard statistics .A total of 183 patients enrolled in the all-document patient set (APS) fulfilled the inclusion criteria and were available for baseline analysis, resulting in identical APS and baseline analysis set (BAS) datasets. Of the 183 patients from BAS, 4 patients withdrew their informed consent, and no post-baseline documented information was available for 3 patients, hence the full analysis set (FAS) is based on the data of the remaining 176 patients and is the population used in this analysis .Most patients (120/176 [68.2%]) had a knee tumor, and 108/176 (61.4%) of the patients were female. The mean (±SD) age at enrollment was 43.5 ± 14.29 years. Baseline demographics are summarized in n = 42) or planned to receive surgery (n = 5), 48 patients (27.4%) received or planned to receive systemic therapy, and 6 patients (3.4%) received (n = 5) or planned to receive radiotherapy  either received (n = 36), followed by systemic therapy , and radiotherapy  were being actively treated at baseline (current treatment), of which 37 (84.1%) received systemic therapy  while the other 7 patients (15.9%) were treated with surgery . Furthern = 47, 1.00 vs. n = 9, 2.86; n = 46, 1.50 vs. n = 9, 3.00; n = 48, 3.0 vs. n = 10, 4.5; n = 48, 0.83 vs. n = 10, 0.74; n = 48, 80.0 vs. n = 10, 67.5; n = 43, 0.57 vs. n = 4, 2.57; n = 44, 1.13 vs. n = 4, 2.63; n = 45, 2.0 vs. n = 4, 3.0; n = 44, 2.0 vs. n = 4, 4.5; n = 46, 80.0 vs. n = 3, 65; This section compares patients who were Off-Treatment at baseline and remained Off-Treatment throughout the 2-year observation period versus patients who changed from Off-Treatment to On-Treatment (systemic treatment or surgery) at years 1 and/or 2. For patients who were Off-Treatment at baseline, numerically favorable scores  were observed in patients who remained Off-Treatment compared with patients who switched to an active treatment strategy at year 1: median BPI Pain Interference  as compared with those who remained on systemic treatment  and 2-year follow-up   to the 2-year visit . EQ-5D index  and 2  to 2 , years 1  and 2  . BPI PaiFrom baseline through the 2-year follow-up visit, patients who remained as wait-and-see and surgery showed an improvement in BPI Pain Interference . No chan28 In previous analyses, the TOPP registry described the impact of TGCT on PROs from a baseline snapshot,25 and subsequently a picture of the treatment journey of D-TGCT patients as a 2-year observational follow-up was reported.26 This analysis is the first to describe the impact of the disease on PROs as a 2-year follow-up based on treatment strategies.TOPP represents the first and largest prospective, international, multicenter disease registry for D-TGCT, and includes 176 patients, demonstrating that conducting collaborative observational studies for a rare tumor is feasible. Due to the rarity of the disease, the current TGCT literature contains predominantly small, retrospective cohort studies, including heterogeneous data.29 Since D-TGCT is a locally aggressive disease affecting large joints and tendon sheaths, it is associated with functional impairment and negatively impacts patients’ QoL.30 Specifically, pain, swelling, stiffness, and reduced range of motion are considered hallmarks of the disease as they are the most common and critical symptoms experienced by patients with TGCT. As disease extent and the type of treatment approach might differently affect physical function and QoL, PRO assessments of disease symptoms, physical function, and HRQoL can serve as a valuable tool to support the relevance of primary endpoints in clinical trials.31Health-related QoL (HRQoL) is a key measure for the effectiveness and cost effectiveness of healthcare interventions and is used to evaluate the impact of the disease on the patients’ QoL assessment; however, HRQoL assessment in sarcoma is challenging due to the diversity of the disease and tumor location.31-34 Specifically, PROMIS-PF and Worst Stiffness scores were included in ENLIVEN to validate reduction in tumor size was meaningful to patients.33 As only changes of ≥3 score units for physical function are deemed meaningful by patients with D-TGCT, only minimal improvement was observed from baseline through the 2-year visit.PRO measurements and QoL evaluation are therefore included as secondary outcome measures in most clinical trials, since it has been demonstrated that improvement of physical function and QoL were important treatment outcomes for patients with D-TGCT.In this prospective analysis, there was a high patient compliance with PRO questionnaire completion. At baseline, the completion rate ranged from 93.8% (Worst Stiffness) to 96.0% , and at the 2-year follow-up visit, most patients (>80%) had completed the questionnaires, demonstrating a high acceptance and interest for the study of this rare disease.15 Numerically improved PRO scores for BPI Pain Interference and BPI Pain Severity as well as for Worst Stiffness at the 2-year visit compared to baseline were observed.As D-TGCT severely affects the patients’ QoL and is responsible for pain and various degrees of limitations sustained over time, this plays a major role in the choice of the treatment strategy. As optimal treatment strategies remain to be elucidated, the multimodal treatment of D-TGCT patients is complex and depends on factors, such as tumor status at the time of assessment, previous treatment history, the clinical background of the treating physician, and information on PROs. At the baseline visit, different treatment approaches, mainly wait-and-see, surgery, or systemic treatment, were undertaken, and the treatment was monitored during the first and the second year of the observation period. For patients who started at baseline with no treatment, remaining Off-Treatment over the 2 years resulted in numerically better PRO scores. Specifically, lower scores were seen for the BPI Pain Interference, BPI Pain Severity, Worst Stiffness, and Worst Pain scales. As certain patients did not develop pain, there was no need to change from Off-Treatment to an active treatment strategy. Only in the instance of pain and discomfort complaints was a change to an On-Treatment strategy needed. For patients who started at baseline with systemic treatment (mainly pexidartinib or imatinib), remaining with systemic treatment over the 2 years resulted in numerically lower BPI Pain Interference and BPI Pain Severity scores, and higher EQ-5D index and EQ-5D VAS scores at year 1 compared with patients who switched treatment strategy. All patients who underwent surgery at baseline had no need for a different treatment within the 2-year follow-up visit; this confirms the disease’s biological behavior, which has a low growth rate and rarely presents with progressive disease and significant complaints within the first 2 years.36 This emphasizes the need for a multidisciplinary approach in which systemic therapy may be a valuable treatment option prior to and/or after surgical resection.37Of note, at baseline, numerically favorable PROs were observed in Off-Treatment patients compared with patients in the systemic treatment and surgery groups. This might reflect a different patient population in terms of size, rate of relapse after previous treatment, or time from diagnosis. In D-TGCT patients, there is always a risk of aggravating symptoms by active treatment , thus remaining Off-Treatment may be beneficial and have a better risk-to-benefit reward regarding PROs. The PROs recapitulate potential symptom triggers for treatment decisions. They also highlight the potential deterioration of the symptom load depending on specific therapeutic interventions, such as repetitive surgery. More specifically, a further deterioration of QoL was seen at 1 year, which was in large part due to D-TGCT treatment . Following systemic treatment, an association between tumor shrinkage and improvement in PROMIS-PF, pain relief, and Worst Stiffness scores have been reported.disease effect associated with D-TGCT, but also the potential treatment effect with lower PRO scores after surgery.Most of the actual D-TGCT treatment was performed during the first year of the observation period, and the decision for a particular treatment was made taking several factors into account, which included the treating physicians’ clinical experience and PRO. PRO analyses demonstrated that D-TGCT severely affects the patients’ QoL and is responsible for pain and various degrees of limitations sustained over time, affecting the choice of the treatment strategy. Further deterioration of QoL was seen after 1 year of follow-up after surgery, which was mainly observed (and possibly caused) by D-TGCT treatment itself . Thus, the PRO measurements illustrate the continued heavy These findings indicate how PROs are relevant to show clinical improvement in patients with D-TGCT and perhaps warrant further assessment in larger patient populations to make treatment decisions in the future. As current literature lacks treatment guidelines and does not present relevant clinical findings that support clinical decision making, creating insight into such important factors can be of great value in optimizing treatment strategies focusing on the most significant individual outcome measure.n = 61/176, 34.7%) were enrolled from 2 orthopedic sites in the Netherlands. The results of this study should be evaluated with care, as they might be prone to potential data bias. There remains a lack of systemic treatment options with only 1 agent approved in the United States and none in Europe. As the majority of the patients remained on the same treatment strategy, there were small numbers in some of the groups pertaining to patients that switched treatment courses. In turn, there is no statistical comparison, with the findings being descriptive.As this project is an observational study, the quality of the collected data has not been 100% verified. Due to the COVID-19 pandemic, the planned on-site monitoring visits in the United States could not be performed. Instead of on-site monitoring visits, more frequent and detailed remote monitoring visits were performed for these sites. Follow-up data for all enrolled patients were regularly monitored remotely, with additional review to minimize potential errors in data acquisition and reporting. The study spanned only 2 years, although it provides an entire overview of the patient journey in a slowly growing disease that can have effects for decades. Both patients in clinical studies and expanded access programs were allowed to enter the study. The study sites are tertiary sarcoma centers that include a difficult-to-treat population, and this study does not allow for analysis of the entire spectrum of TGCT. One-third of the patients , illustrating the need for novel approaches in this population. The findings from the current analysis emphasize the relevance of PROs in TGCT trials. As novel treatments for D-TGCT are being actively studied, this analysis represents a benchmark and should be considered in future studies, including CSF1R inhibitors and surgical trials.oyad011_suppl_Supplementary_Figure_S1Click here for additional data file.oyad011_suppl_Supplementary_Figure_S2Click here for additional data file.oyad011_suppl_Supplementary_Figure_S3Click here for additional data file.oyad011_suppl_Supplementary_Figure_S4Click here for additional data file.oyad011_suppl_Supplementary_Figure_S5Click here for additional data file.oyad011_suppl_Supplementary_Figure_S6Click here for additional data file.oyad011_suppl_Supplementary_Figure_S7Click here for additional data file.oyad011_suppl_Supplementary_Figure_S8Click here for additional data file.oyad011_suppl_Supplementary_Figure_S9Click here for additional data file.oyad011_suppl_Supplementary_Table_S1Click here for additional data file.


The work is expected to provide some guidance on specific intervention policies in relation to the types of indoor environments.There exist various vertical temperature gradients in different-type buildings. A holistic understanding of the impact of different temperature-stratified indoor environments on infection risk is necessary. In this work, the airborne transmission risk of SARS-CoV-2 in different thermally stratified indoor environments is assessed using our previously developed airborne infection risk model. Results show that the vertical temperature gradients in office building, hospital, classroom, etc. are within the range of −0.34 to 3.26 °C/m. In large space such as coach station, airport terminal, and sport hall, the average temperature gradient ranges within 0.13–2.38 °C/m in occupied zone (0–3 m); in ice rink with special requirements of indoor environment, the temperature gradient is higher than those in the above indoor spaces. The existence of temperature gradients causes multi-peaks of the transmission risk of SARS-CoV-2 with distancing, and our results show that in office, hospital ward and classroom, the second peak of the transmission risk is higher than 10the Appendix is available in the online version of this article at 10.1007/s12273-023-1021-5. Impact of thermal stratification on airborne transmission risk of SARS-CoV-2 in various indoor environments